Project Abstract Type 2 diabetes is the most common metabolic disease and affects nearly 10% of the American population. Given its close association with obesity, not only is the prevalence expected to increase in the future, but individuals with the disease are at a higher risk for developing severe health complications, including cardiovascular disease, which remains the leading cause of death for diabetic patients. Therefore, it is imperative that better treatment strategies are developed to manage the progression of the disease and its associated health risks. Many factors, both genetic and environmental, contribute to the manifestation of type 2 diabetes; however, the development of peripheral insulin resistance remains the strongest etiological predictor of disease progression. An abundance of scientific evidence suggests that chronic inflammation, particularly in the setting of obesity, promotes cellular insulin resistance. Specifically, pro-inflammatory cytokines are elevated in metabolic diseases and can inhibit insulin-dependent signaling pathways in tissue sites such as the liver. Recently, a unique link between a family of proteins known as the TNF receptor-associated factors (TRAFs) and the development of insulin resistance has been developed. TRAF proteins are key mediators in cytokine signaling, but their role in the liver, particularly hepatocytes, are not well defined. Preliminary data suggests that one specific member, TRAF3, negatively regulates insulin sensitivity in primary hepatocytes as well as in transgenic mouse models by promoting the ubiquitination of insulin receptor substrates 1 and 2 (IRS1/2), which function as important adaptor proteins that facilitate insulin signaling transduction pathways in target cells. Therefore, the working hypothesis of this proposal is that hepatic TRAF3 expression directly promotes insulin resistance in the liver through altered ubiquitination of IRS1/2 adaptor proteins. To test this hypothesis, two aims have been developed. Aim 1 seeks to determine the mechanisms that drive hepatic TRAF3 induction in obesity. Preliminary data suggests that TRAF3 is up-regulated in mouse models of obesity. To determine the mechanisms by which this occurs, a variety of approaches, conducted in primary hepatocytes and mouse models, will be employed to investigate the roles of cytokines, free fatty acids, and lipopolysaccharide in promoting TRAF3 up-regulation in a Toll-like and TNF receptor-dependent fashion. Aim 2 seeks to determine the molecular mechanisms by which TRAF3 promotes insulin resistance. Specifically, TRAF3's role as a ubiquitin ligase will be the focus by using transgenic mouse strains, molecular biology and analytical chemistry techniques, such as mass spectrometry, to identify the sites of IRS1/2 ubiquitination and its functional outcome in the liver. Together, these studies will advance the scientific understanding of how inflammation and IR are linked, as well as provide novel approaches and strategies to treat type 2 diabetes and its associated complications.